Retinoic acid (RA) signaling is central to the spermatogonial differentiation. Despite this fact, the molecular mechanisms that govern the spermatogonial differentiation remain poorly understood. While several groups are studying RA-mediated cellular transcriptome changes during spermatogonial differentiation, the effect of RA on gene expression at the posttranscriptional level is yet to be elucidated. MicroRNAs (miRNAs) are a new class of short non-coding RNA genes that function as sequence-specific posttranscriptional regulators of gene expression. It has become apparent that miRNAs play important roles during spermatogenesis. However, it is unclear whether miRNAs can be regulated by RA signaling and could thereby contribute to spermatogonial differentiation. Our long-term goal is to identify the essential networks involved in spermatogonial differentiation. Our central hypothesis is that RA regulates miRNAs expression and these miRNAs in turn modulate expression of key genes encoding protein responsible for spermatogonial differentiation. In this R21 application, we propose to determine how RA-regulated miRNAs potentially contribute to spermatogonial differentiation. Specifically, in Aim 1 we hypothesize that expression of a subset of miRNAs is regulated by RA signaling and we propose to identify the RA-regulated miRNAs during spermatogonial differentiation. In Aim 2 we hypothesize that RA-regulated miRNAs modulate expression of key downstream proteins to enhance the effect of RA on spermatogonia differentiation and we propose to establish the potential functional roles of selected RA-regulated miRNAs in spermatogonial differentiation. Completion of the proposed research will help identify regulatory networks that control spermatogonial differentiation. A better understanding of spermatogonial differentiation has important implications for male reproduction and contraception. PUBLIC HEALTH RELEVANCE: Male factor infertility occurs in about 10% of men, although the etiology of impaired spermatogenesis is rarely identified. Retinoic acid (RA) is critical for spermatogonial differentiation, an initial step of spermatogenesis. The purpose of this proposal is to explore the role of RA-targeted microRNAs in the regulation of spermatogonial differentiation, which has important implications for retinoid biology and male reproduction.